Influenza is a major cause of morbidity and mortality around the world. In the United States, seasonal epidemics of flu occur from late fall to early spring affecting all age groups but with especially high rates of infection in children. Serious illness and death are consequences of influenza infection among the elderly, young children, and individuals with other illnesses that predispose them form complication of influenza. Vaccination is an effective way to counter influenza infection and illness, but several scientific and technical considerations can negatively impact the current strategies for vaccination against this virus. Because of the antigenic drift for which the influenza virus has a considerable propensity, the strain of virus included in each season's vaccine is frequently change to reflect the current circulating virus. The issue of antigenic drift is perhaps the most significant impediment to the production of a "universal" or broadly cross- protective vaccine to influenza. Attempts to overcome the diversity of influenza strains have been the focus of many efforts. In this proposal we will attempt to solve a particular problem that has been identified by the FDA related to the influenza B virus component of the seasonal vaccine. Unlike influenza A viruses, influenza B infects almost exclusively humans and has been responsible for severe epidemics with high rates of hospitalization for young children. Two lineages of influenza B have been recognized for many years. Although there is antigenic drift within each lineage, the sequence diversity is relatively limited. However, it has proven to be difficult to forecast which lineage will dominate a given flu season and the FDA has suggested that both strains be included in the seasonal vaccine, thus requiring a quadrivalent vaccine to be developed. A simpler solution would be to create an influenza B immunogen that induces cross-protective immunity to both lineages. We propose to use a directed molecular evolution approach to address this problem. Many variants of influenza B hemagglutinin will be created using in vitro DNA recombination of the genes encoding the various hemagglutinin proteins that have been used in previous vaccines. Immunization of mice with many of these variants and analysis of the neutralizing capability of the resulting serum will be used to screen for immunogens that meet the desired criteria. Although we are cognizant of the fact that changes in influenza hemagglutinin sequences arise largely through point mutation, several possibilities support the idea that creation of novel diversity by recombination can improve immunogenicity. Recombination might combine epitopes from different viral variants or expose conserved neutralizing epitopes into one immunogen capable of eliciting broader protective responses. Our primary objective is to have a fully cross-reactive neutralizing response from a novel influenza B immunogen;however, a secondary objective is to broaden the response to cover all viruses of one lineage. PUBLIC HEALTH RELEVANCE: The annual influenza season is responsible for considerable sickness and death, especially among the more vulnerable members of the population. Since flu strains change from year to year, a vaccine that acts broadly would be of great value. We propose to create an improved version of one of the components of the seasonal vaccine that can provide better protection against some of the flu viruses.